Two billion people are currently infected with the infectious organism, M tuberculosis, which causes tuberculosis. Understanding TB pathogenicity is critical for the development of more effective treatment and vaccines that could change the global emergency. This research proposal will use novel genetic tools to create specific TB mutants that will be used to elucidate the role of HtrA-like serine proteases in M tuberculosis pathogenicity. HtrA serine proteases have been shown to be associated with virulence in many organisms including Salmonella, Yersinia, and Brucella, and are induced by exposure to heat shock and oxidative and osmotic stresses. By using a novel phage delivery system, allelic exchange will be performed in TB to create mutants with deletions in the HtrA-like serine protease genes: htrA, pepA, and Rv0983. These 3 TB mutants will be characterized in vitro for their growth characteristics, thermosensitivity, and ability to survive in cultured macrophages. TB serine protease mutants will also be characterized in vivo by infecting C57BL/6 mice and BALB/c SCID mice to determine if mutants are attenuated in the murine model. HtrA mutants in other organisms have been shown to act as vaccines by protecting mice against a lethal challenge with the wildtype organism. BALB/c mice will be vaccinated with TB serine protease mutants and challenged with a virulent wildtype strain of TB.